Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

Glycine receptors are chloride-permeable, ligand-gated ion channels and contribute to the inhibition of neuronal firing in the central nervous system or to facilitation of neurotransmitter release if expressed at presynaptic sites. Recent structure-function studies have provided detailed insights into the mechanisms of channel gating, desensitization, and ion permeation. However, most of the work has focused only on comparing a few isoforms, and among studies, different cellular expression systems were used. Here, we performed a series of experiments using recombinantly expressed homomeric and heteromeric glycine receptor channels, including their splice variants, in the same cellular expression system to investigate and compare their electrophysiological properties. Our data show that the current-voltage relationships of homomeric channels formed by the ␣2 or ␣3 subunits change upon receptor desensitization from a linear to an inwardly rectifying shape, in contrast to their heteromeric counterparts. The results demonstrate that inward rectification depends on a single amino acid (Ala 254 ) at the inner pore mouth of the channels and is closely linked to chloride permeation. We also show that the current-voltage relationships of glycine-evoked currents in primary hippocampal neurons are inwardly rectifying upon desensitization. Thus, the alanine residue Ala 254 determines voltage-dependent rectification upon receptor desensitization and reveals a physio-molecular signature of homomeric glycine receptor channels, which provides unprecedented opportunities for the identification of these channels at the single cell level.

Section: -H)supporting

confidence: 81%

Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels

Raltschev

Hetsch

Winkelmann

et al. 2016

“…M1, however, played an important role for expressing these extracellular domain nAChRs. These results, with the properties of extracellular domain ␣7 nAChRs (33,34) and evidence of oligomerization of extracellular domains from ␣1 and ␦ subunits (31,32), from extracellular domains of muscle-type subunits (35), from chimeric ␣7/AChBP subunits (26), from glycine receptor subunits (68,69), and from GABA A receptor subunits (70), suggest that producing extracellular domain receptors might be possible for many subunits throughout the nicotinoid family. Expression of extracellular domain nicotinoid receptors with high structural fidelity, however, might be more feasible with M1 included in the subunit design than without M1.…”

Section: Discussionmentioning

confidence: 84%

Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

Person

Bills

Liu

et al. 2005

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of ␣7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of ␣4 and ␤2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with ␣4␤2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain ␣4␤2 nAChRs formed from subunits with M1 were nearly identical to affinities of ␣4␤2 nAChRs when measured with [ 3 H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of ␣4␤2 nAChRs. In contrast, [3 H]epibatidine binding was not detected from the extracellular domain ␣4 and ␤2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both ␣4 and ␤2 play an important role for efficient expression of extracellular domain ␣4␤2 nAChRs that are high fidelity structural models of full-length ␣4␤2 nAChRs. Nicotinic acetylcholine receptors (nAChRs)2 are ligand-gated ion channels expressed mainly in the nervous system and at the neuromuscular junction (1-3), although they also are expressed elsewhere (4). They are members of the superfamily of nicotinoid receptors, which includes ␥-aminobutyric acid (GABA) type A and C, glycine, and serotonin 5-HT3 receptors. They contain five homologous subunits, with subunits designated ␣1-␣10, ␤1-␤4, ␦, ␥, and ⑀. Each subunit is an integral membrane protein with a large, ligand-binding N-terminal extracellular domain, four transmembrane domains (M1-M4), and a cytoplasmic loop between M3 and M4. The study of structure and dynamics of nAChRs has been motivated by their roles in normal and pathologic neurophysiology, including roles in addiction, neurodegeneration, epilepsy, myasthenia gravis, and congenital myasthenic syndromes (5).Knowledge of the structure of nAChRs has come from biochemical, electrophysiological, and imaging methods (6 -8). These methods, however, do not provide comprehensive structure at atomic resolution. A significant advance toward such information has come recently from the crystallographic structure of acetylcholine-binding protein (AChBP), a water-soluble pentamer that has been the basis for homology modeling of the extracellular...

“…Bacterial Protein Expression and Refolding-Recombinant expression of the isolated GlyR␣1 ECD constructs and refolding under oxidative conditions (using CuCl 2 ) was carried out essentially as described (15). Deviating from this protocol, RosettaGami2 cells (EMD) were used for expression and affinity purification was omitted.…”

Section: Methodsmentioning

confidence: 99%

“…3 H]strychnine and [ 3 H]glycine (PerkinElmer Life Sciences) to refolded proteins was performed as described (15). To achieve retention of the refolded protein on GF/C filters, it was precipitated with an equal volume of 15% PEG400 in binding buffer (25 mM potassium P i , pH 7.4, 200 mM KCl).…”

Section: H]ligand Binding Assaysbinding Of [mentioning

confidence: 99%

See 1 more Smart Citation

Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

Vogel

Kluck

Melzer

et al. 2009

Self Cite

The strychnine-sensitive glycine receptor (GlyR) is a ligandgated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cysloop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChR␣1 ECD, we generated a model of the human GlyR␣1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyR␣1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.The glycine receptor (GlyR) 2 is a ligand-gated ion channel that mediates fast neuronal inhibition predominantly in the spinal cord and brain stem (1). In the mammalian central nervous system, five gene variants of homologous GlyR subunits have been identified (2), four belong to the ␣-type (␣1-␣4) and one to the ␤-type. The GlyR complex presents as a rosette-like assembly of five subunit polypeptides surrounding a central anion pore. During development of the spinal cord, embryonic ␣2-homopentamers are largely replaced by adult heteromeric isoforms (3), composed of 3 ␤-and 2 ␣-subunits (4).The GlyR belongs to the superfamily of cysteine loop (Cysloop) receptors, which also comprises GABA A/C (␥-aminobutyric acid) receptors, nicotinic acetylcholine receptors (nAChR), and 5-hydroxytryptamine type 3 receptors. Structural predictions for Cys-loop receptors rely on sequence-based predictions and mutational studies. Crystal structures of the Lymnaea stagnalis acetylcholine-binding protein (AChBP), the Torpedo marmorata nAChR, the extracellular domain (ECD) of the murine nAChR␣1, and prokaryotic proton-gated channels indicate a common, conserved protein fold (5-8). Accordingly, each subunit of a Cys-loop receptor comprises a large amino-terminal ECD adopting a twisted ␤-sandwich structure that pr...